Method and apparatus for tiling light sheet selective plane illumination microscopy with real-time optimized light sheet

ABSTRACT

Disclosed are a method and apparatus for tiling light sheet selective plane illumination microscopy (TLS-SPIM) with real-time light sheet optimization. The method and apparatus image multi-cellular specimens in 3D, with an improved 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. A 3D live imaging ability is provided of the TLS-SPIM by real-time imaging cellular and sub-cellular behaviors.

PRIORITY

This application claims priority to Provisional Patent Applications No.62/119,552, 62/150,531, 62/198,969 and 62/220,572 filed Feb. 23, 2015,Apr. 21, 2015, Jul. 30, 2015 and Sep. 18, 2015, respectively, thecontent of each of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates generally a fluorescence microscopy technique tothree dimensional (3D) imaging of live biological specimens in realtime.

Cells live in 3D environments. A more accurate understanding of cellbehaviors and cell-cell interactions can be obtained by studying cellsin their native, multi-cellular environments than when cultured onsubstrates. In a multi-cellular organism, the intracellular activitiesof a cell affect not only the cell itself but also its neighbor cells,and the cellular behavior of a cell results from both its ownintracellular activities and its interactions with other cells. In otherwords, cell behaviors in a multi-cellular process are caused by theintracellular activities of all cells involved in the process. Thus, inorder to understand cell behaviors in a multi-cellular process, it isnecessary to study all involved cells at sub-cellular level to acquirethe dynamic information of their intracellular activities, by which theunderlying connections between cellular behaviors and intracellularactivities of the involved cells can be revealed. For this reason,fluorescence imaging techniques that allow low-invasive 3D imaging ofmulti-cellular specimens with high spatial and temporal resolution arerequired.

Multi-cellular specimens are difficult to image in 3D because of theirsize and complexity accompanied with optical aberrations and lightscattering. Both high 3D spatial resolution and good optical sectioningcapability are required in a large field of view (FOV) in order tovisualize the specimen with sub-cellular, or even cellular structuraldetails. Meanwhile, the photobleaching and photodamage must be lowenough to allow live imaging at needed speed for a certain period oftime. Therefore, selective plane illumination microscopy (SPIM) isgetting increasing attention for its advanced 3D live imaging ability[1-4]. Generally, the latest SPIM techniques allow low-invasive,high-speed 3D imaging of either single cell specimens with sub-cellularlevel, submicron 3D spatial resolution [5-8], or multi-cellularspecimens with cellular level spatial resolution of a few microns[9-12], although the actual performance varies depending on the sampleand fluorophore. However, imaging multi-cellular specimens in 3D withsub-cellular spatial resolution remains challenging despite the progressthat has been made in SPIM development.

SPIM obtains 3D imaging ability by confining the illumination light nearthe detection focal plane with a light sheet. With a given detectionnumerical aperture (NA), its 3D imaging ability, including the spatialresolution, optical sectioning capability, field of view (FOV),photobleaching and photodamage, is mainly determined by the intensityprofile of the light sheet [13, 14]. A uniformly thin and large lightsheet is therefore required in SPIM to maximize its imaging ability, andthe generation of such a light sheet has been a major focus of SPIMdevelopment [5-7, 15, 16]. Although different methods have beendeveloped, none of them are ideal. Essentially, every light sheetbalances the properties of light sheet thickness, the illumination lightconfinement, and the light sheet size differently, which results indifferent 3D spatial resolution, optical sectioning capability and FOV,respectively [13, 14]. Nevertheless, it becomes extremely difficult tobalance these properties as the FOV increases to image multi-cellularspecimens of dozens of microns or larger, since an ideal light sheetthat has thin thickness, good light confinement and a large size at thesame time does not exist due to the diffraction of light. Either thespatial resolution, optical sectioning capability, or both must besacrificed to reach a larger FOV because the light sheet either becomesthicker, or the excitation light is less confined as its size increases.Therefore, the tradeoff between the spatial resolution, opticalsectioning capability and FOV sets a fundamental limit on conventionalSPIM, and a key problem of imaging multi-cellular specimens withsub-cellular spatial resolution using SPIM turns out to be how toincrease the FOV without losing the spatial resolution and opticalsectioning capability. Different approaches other than finding a perfectlight sheet must be made.

Multiview SPIM is a different approach that works in two ways to improvethe 3D imaging ability of SPIM on multi-cellular specimens. First, bysending the excitation light sheet and collecting the fluorescencesignal from different directions, the final image is less affected bythe optical aberration and light scattering introduced by the sample[9-12]. Next, 3D images taken from different directions can be fusedtogether with similar methods used in tomography, by which the 3Dspatial resolution can exceed the resolution limit set up by the lightsheet in theory [1, 8, 12]. Nevertheless, the improvement in resolutionby this approach relies on the number of different view directions, andthe spatial resolution and the signal to noise ratio (SNR) of the 3Dimages taken in these different views. In multiview SPIM, the number ofview directions is limited to two (lateral and axial) without rotatingthe sample, while the spatial resolution and SNR of the 3D image takenin each view are still strictly constrained by the light sheet used totake the image. Therefore, multiview SPIM cannot bypass the fundamentaltradeoff of SPIM set up by the light sheet. Furthermore, high-speedsub-cellular dynamics, the optical aberration and light scatteringintroduced by both the sample and the agarose gel usually used to mountthe sample make it more difficult to fuse different views accuratelywith high spatial resolution. Consequently, multiview SPIM is suited forimaging relatively large and bright multi-cellular specimen withcellular level spatial resolution. Imaging multi-cellular specimens withsub-cellular spatial resolution remains a problem with multiview SPIM.

The nonexistence of a perfect light sheet not only limits the 3D imagingability of SPIM, but also creates a severe yet underappreciatedpractical problem. Optimization of the light sheet, including its typeand dimensions, is required case by case in SPIM depending on the sampleto be imaged and the biological question to be studied, as differentlight sheets balance the 3D imaging ability of SPIM differently [14].The light sheet is usually determined based on the prior knowledge ofthe specimen and the biological process to be imaged, and it remains thesame during the entire imaging process because a realignment of themicroscope, that often takes hours even for SPIM experts, is usuallyrequired to change the light sheet. It is not only extremelyinconvenient, but also prevents SPIM from adapting itself to reach theoptimal imaging performance, because different organelles have differentstructures and dynamics, and both of these can change in live imaging.Instead, the optimization of SPIM imaging performance would become aclosed-loop process if the light sheet could be adjusted in real-timeusing the immediate imaging result as feedback. More importantly, theunderstanding of a biological process relies on the spatial and temporalinformation that can be obtained from the process, whereas an idealmethod that is capable of acquiring both with a high resolution is oftenunavailable, especially for multi-cellular specimens. The only optionunder such circumstances is to compromise either the spatial or thetemporal resolution strategically, and only acquire the necessaryinformation needed to understand a biological process. Such ability andflexibility to compromise can only be obtained by adjusting the lightsheet in real-time in SPIM. Thus, be able to optimize the excitationlight sheet in real-time is another key to improving the 3D live imagingability of SPIM on multi-cellular specimens.

SUMMARY OF THE INVENTION

To overcome shortcoming of conventional SPIM in imaging largemulticellular specimens, provided is a method of three-dimensional (3D)multi-cellular specimen imaging, with the method including using areal-time adjusted, tiling light sheet (TLS) selective planeillumination microcopy, wherein both high spatial resolution and goodoptical sectioning capability are maintained within a large field ofview (FOV), and wherein the FOV is larger than the tiling light sheet.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following detailed description of preferred embodiments is made withreference to the accompanying drawings. In describing the invention,explanation of related functions or constructions known in the art isomitted for the sake of clarity in understanding the concept of theinvention and to avoid obscuring the description of the invention withunnecessary details.

Recently, we described a tiling light sheet SPIM technique (TLS-SPIM) toovercome the SPIM tradeoff between the spatial resolution, opticalsectioning capability and FOV by tiling the excitation light sheet [17].Instead of using a large light sheet to illuminate the entire FOVsimultaneously, a small but thin light sheet is tiled quickly within theimage plane to illuminate the whole FOV, while only the fluorescencesignal generated at center of each tiled light sheet is used in thefinal image. As a result, both high spatial resolution and good opticalsectioning capability are maintained within a FOV that is much largerthan the light sheet itself. Potentially, multi-cellular specimens couldbe imaged by TLS-SPIM with either higher spatial resolution, betteroptical sectioning capability or both compared to regular SPIM. BesidesTLS-SPIM, several other methods were also developed to increase the FOVof SPIM with different approaches [18-20]. Unfortunately, the imagingability of these methods are limited by using either two-photonexcitation or optical components with slow dynamic performance, whilenone of these methods can implement all of the latest SPIM light sheetsand optimize the light sheet in real-time.

Convinced by the imaging ability of TLS-SPIM observed in our previousresearch [17], we continued to develop and explore the potential of thismethod to facilitate cell behavior study in multi-cellular specimens. Wepresent TLS-SPIM with real-time light sheet optimization to address theabove problems that limit SPIM in imaging multi-cellular specimens withsub-cellular spatial resolution. We extended the 3D live imaging abilityof TLS-SPIM by enabling the implementation, tiling and real-timeoptimization of the latest SPIM light sheets in TLS-SPIM, including theGaussian, Bessel and Lattice light sheet. We compared the 3D imagingability of TLS-SPIM in resolving complex structures with regular SPIMmethods and verified its 3D live imaging performance on differentmulti-cellular specimens. In addition, we introduce a methodology tostudy cell behaviors in multi-cellular specimens using TLS-SPIM, whichis to image intracellular activities of the cells involved in amulti-cellular process, identify their intrinsic connections with thecellular behaviors, and determine their impact on the multi-cellularprocess. We demonstrate the methodology using C. elegans embryoleft-right symmetry breaking behavior as an example.

Our new TLS-SPIM microscope was designed to satisfy two requirements.First, the microscope can implement the latest SPIM light sheets to usetheir advantages in different applications, and the light sheet can beadjusted quickly to optimize the TLS-SPIM imaging performance inreal-time. Next, the light sheet can be tiled rapidly within thedetection image plane to enlarge the FOV without affecting the spatialresolution and optical sectioning capability. To achieve these goals,two binary spatial light modulators (SLM) are implemented in sequence inour TLS-SPIM microscope to modulate the excitation light. The two SLMsare conjugated to the image focal plane and the rear pupil of theexcitation objective, respectively. Either a Gaussian light sheet, aBessel light sheet or a Lattice light sheet can be used in themicroscope, and it takes less than a millisecond to either tile orchange the light sheet by applying different binary phase maps to theSLMs.

To examine the ability of TLS-SPIM in resolving complex structures, weimaged a C. elegans embryo (OD95) expressing GFP::PLC∂PH (membrane) andH2B::mCherry (nucleus) every 30 minutes for ˜5 hours, from ˜50 cellstage to comma stage until the muscle cells were functioning. A ˜0.7 μmthick, ˜10 μm long Bessel light sheet, generated by scanning a singleBessel beam, was tiled at three positions at 10 μm intervals to imagethe embryo. The nearly isotropic spatial resolution (˜320 nm lateral,˜460 nm axial) and good optical sectioning capability enabled theidentification of individual cells at most developmental stages. Suchimaging ability will allow the study of morphogenesis at the necessarysub-cellular resolution through later stages of embryonic developmentthan was previously possible via other techniques. Specifically, we showthat it is difficult to obtain similar results by conventional SPIM witheither Gaussian, Bessel or Lattice light sheets, as the light sheet mustbe thicker or the illumination light must be less confined to image thesame FOV in regular SPIM. The comparison also shows that the imagingperformance of different light sheets varies from sample to sample dueto the different sample properties. Instead of sticking to a particularlight sheet or method, a more suitable light sheet, including the type,dimensions, tiling number and tiling positions, can always beimplemented in TLS-SPIM in real-time with the immediate feedback fromthe imaging result.

To demonstrate the 3D imaging ability of TLS-SPIM on larger specimens,we imaged the tailbud of a ˜15 hpf nuclei-labeled zebrafish embryo inthe next. A ˜1.2 μm thick, ˜30 μm long Bessel light sheet, whichprovides ˜320 nm lateral resolution, ˜750 nm axial resolution, was tiledat nine positions at ˜25 μm intervals to image a 0.2 mm×0.2 mm volume.Most cell nuclei can be identified and examined with sub-cellularstructural details. Again, the structure was better resolved by TLS-SPIMas compared to regular SPIM attributed to the usage of a thinner lightsheet with better light confinement. Nevertheless, as can be observedfrom the result, the 3D imaging ability of TLS-SPIM on multi-cellularspecimens is still limited by optical aberration and light scatteringthat affect all light microscopy techniques.

Due to the light sheet tiling in TLS-SPIM, each image plane isilluminated multiple times equivalent to the number of tiling positions,which raises concerns of slower imaging speed and higher photobleachingand photodamage. In regular SPIM, either the spatial resolution, opticalsectioning capability or both must be traded for a larger FOV, while inTLS-SPIM, the temporal resolution can be traded as a substitute for FOVby tiling the light sheet. The decision of whether to sacrifice thetemporal resolution, and how much to sacrifice will depend on thebiological question to be answered. In addition, the problems caused bylight sheet tiling are fully predictable and controllable by limitingthe number of tiling positions.

To evaluate the live imaging performance of TLS-SPIM, we imagedendogenously labeled myosin II particle activities in a live C. elegansembryo (LP162) every 8.5 seconds for 150 time points. A Bessel lightsheet, generated by scanning an incoherent seven Bessel beam array, wastiled at three positions to image the embryo. Both the spatial andtemporal resolution of our results are sufficient to track and analyzethe myosin II particle movements in 3D, by which the behavior of theactomyosin network and its impact on embryonic cells can be studied. Inanother example, we imaged a C. elegans embryo (OD95) with the samelight sheet every 30 seconds in two channels for 167 time points. Bothhigh spatial resolution and good SNR were maintained through theprocess, and the embryo hatched successfully in the end. The resultshows comparable spatial resolution, SNR, imaging speed and much longerimaging time compared to the results obtained from similar specimensimaged by Bessel SPIM and Lattice light sheet microscopy in previouspublications [6, 7]. The result allows us to extract the dynamicinformation of every individual cell and analyze their relationship overdevelopmental time, facilitating both visualization and understanding ofcell behaviors as embryonic development progresses. Clearly, TLS-SPIMperforms well for 3D live imaging in terms of the spatial resolution,SNR, imaging speed, photobleaching and photodamage when the tilingnumber is kept low.

A new methodology can be established to study cellular behaviors inmulti-cellular specimens with the dynamic information of intracellularactivities acquired by TLS-SPIM. One area of active interest has beenthe identification of the left-right symmetry breaking mechanism of C.elegans embryos at the 4 to 6 cell stage [21-24]. TLS-SPIM can providehigh spatial and temporal resolution information of differentintracellular activities involved in the process, such as the actomyosinnetwork behavior and the deformation of each individual cell,implicating the mechanical mechanism that drives symmetry breaking. Weidentified three intracellular activities that could have majorcontributions to the symmetry breaking behavior mechanically. First, thetorque generated by the unaligned contractile rings of the ABa and ABpcells during mitosis could shear the ABar and ABpr to ABal and ABplcells. Second, the simultaneous relative rotation of their daughtercells, ABal to ABar and ABpl to ABpr, could be caused by the torquegenerated by the counter flow of asymmetrically distributed myosin IIparticles on the contractile ring of ABa cell, which is consistent withprevious observation [23]. Third, the retreating of the EMS cell,entering mitosis afterwards, could assist the symmetry breaking behaviorand the positioning of all daughter cells by pulling the neighbor cells.Our observations not only provide evidence to verify some of theprevious hypothesis [23, 24], but also suggest new mechanism to explainthe behavior. Based on these observations, more specific experiments canbe designed, quantitative image analysis tools and mechanical modelingmethods can be introduced to analyze and understand the biochemical andbiophysical mechanism of the symmetry breaking behavior. Asdemonstrated, with the advanced 3D live imaging ability of TLS-SPIM,instead of treating different cell behaviors isolatedly, a methodologyof imaging and analyzing intracellular activities of different cells andfinding their intrinsic connections can be applied to study cellbehaviors in multi-cellular specimens.

The methodology can certainly be applied to later stage C. elegansembryos and other multi-cellular specimens with the 3D imaging abilityof TLS-SPIM. For instance, by examining all cells of the same embryo attwo time points of 1.5 minute apart at the 8 to 12 cell stage, wenoticed that both the shape and position of the C cell, E cell and MScell changed following the mitosis of the ABal, ABar, ABpl and ABprcells, that divided almost synchronously. The positions of ABal, ABar,ABpl and ABpr daughter cells also seem affected by the synchronizedmitotic processes of these cells. The consistent observations ofaggressive cell position rearrangement during synchronized mitoticprocesses through the embryo development also suggest that thecontractile rings of synchronized mitotic cells play an important rolein cell positioning and morphogenesis of early stage C. elegans embryos.More imaging and quantitative analysis on different intracellularactivities that are related to force generation are required tounderstand these behaviors completely.

TLS-SPIM requires more tiling positions to further increase the FOV andmaintain the same spatial resolution and optical sectioning capability,making it less ideal for high resolution live imaging of largemulti-cellular specimens. As no imaging technique can achieve submicron3D spatial resolution and high imaging speed simultaneously on specimensof hundreds of microns or larger, we developed a compromised solution tohandle this problem, which is to acquire the spatial and temporalinformation of a biological process separately, namely, alternating the3D imaging between a high spatial resolution, low imaging speed mode anda low spatial resolution but high imaging speed mode. In this way, theresults acquired in different modes could still provide sufficientspatial and temporal information to understand the imaged process, aslong as the switching between different modes is fast enough. OurTLS-SPIM microscope enables a quick switch by adjusting the excitationlight sheet in less than a millisecond, realized by changing the phasemaps applied to the binary SLMs used in the microscope. This aspect wasdemonstrated by imaging cell migration of mosaically labeled mesodermalcells expressing a GFP-tagged membrane marker in the tailbud of a ˜15hpf zebrafish embryo. The region of interest (80 μm×80 μm×50 μm) wasimaged with ˜320 nm lateral resolution, ˜750 nm axial resolution every˜9 seconds, and with ˜320 nm lateral resolution, ˜450 nm axialresolution every minute alternately. By this method, both the 3Dstructure and the dynamics of the migrating cells can be clearlyvisualized. A notable difference between the imaged cells and cellsmigrating on 2D substrates is the absence of membrane ruffling behavior,despite the similar lamellar protrusions [7], which could be caused bymore strict constraints on cell membrane by neighbor cells inmulti-cellular specimens. A reasonable understanding of a biologicalprocess can still be obtained by compromising the imaging abilityflexibly when an ideal method is not available. Such ability isespecially valuable in imaging large multi-cellular specimens as theyare more difficult to image and the cellular behaviors are morecomplicated in such specimens.

TLS-SPIM with real-time light sheet optimization not only offersadvanced 3D live imaging ability, allows new methodology to be appliedto understand cell behaviors in multi-cellular specimens, but alsoenables the delivery of such imaging ability and analysis method togeneral SPIM users with its special instrument design, as there is nohardware change required to operate the microscope after the initialalignment, including the light sheet optimization, tiling, and imagingmode switching. A deep understanding on TLS-SPIM technique itself is notnecessary for general users to use this technique, the most suitableimaging condition, including the light sheet and its dimensions, tilingnumbers, positions and imaging modes can be determined solely based onthe sample features, desired spatial resolution, SNR, imaging speed, FOVand the acquired images. The phase maps to be applied to the SLMs can becalculated automatically and applied accordingly. On the other hand,TLS-SPIM can take the advantages of most previously developed SPIMtechniques rather than conflict with any of them, such as the differentlight sheets and the multiview SPIM configuration. Altogether, TLS-SPIMwith real-time light sheet optimization will make SPIM much morefeasible and favorable than it used to be to general biologists.

Two perceived problems are still limiting the 3D imaging ability andapplication of SPIM. First, SPIM is still limited by optical aberrationand light scatting as other optical imaging techniques, and the problemis doubled in SPIM due to the separated illumination and detection. Aneffective combination of SPIM and adaptive optics without heavilyaffecting its live imaging performance is desired. After all, how wellSPIM works relies on how well the excitation light is confined. Next,SPIM is unique due to the enormous spatial and temporal information thatcan be collected with it. However, appropriate quantitative imageanalysis tools are not yet widely available to general users to makemost of the collected information useful. On the other hand, thedevelopment of appropriate image analysis tools itself faces manychallenges at the same time, because of the huge variety of thebiological questions, the imaging ability difference of the differentSPIM techniques, and the lack of clear indications of what kind ofquantitative information is desired and how the information can be used.The gap between biologists, physicists, and computer scientists must befilled to have this problem solved. Ultimately, moving cell biologyresearch from 2D to 3D is inspiring but still challenging. The solutionsto the above problems will perhaps determine how much impact SPIM canmake in accelerating the transition.

Methods

Design Considerations

The Key Tradeoff in SPIM:

Axial resolution, optical sectioning capability and FOV are the threekey factors affecting the 3D imaging ability of SPIM. These factors areonly determined by the intensity profile of the excitation light sheetin regular SPIM with a given detection NA. The axial resolution,defining the smallest axial separation SPIM can resolve in theory,relies on the thinnest axial component of the excitation light sheet inreal space, which corresponds to the highest axial frequency of thelight sheet OTF in frequency space. The optical sectioning capabilitymeasures the ability of SPIM to suppress out-of-focus fluorescencebackground. It determines whether the theoretical spatial resolution,especially the axial resolution, can be achieved with high enough SNR inpractice. The optical sectioning capability of SPIM depends on thepercentage of the illumination light confined within the detection depthof focus in real space, and it is also reflected by how quickly thelight sheet OTF amplitude rolls off to zero along the axial direction infrequency space [13, 14].

Aside from the excellent axial resolution and optical sectioningcapability of SPIM, FOV is a major limiting factor of SPIM due to theoptical configuration of SPIM. In general, it is limited by the lengthof the excitation light sheet along the light propagation direction,where the light sheet intensity profile remains uniform. For thisreason, the light sheet length must be increased to enlarge the SPIM FOVfor large specimens. However, no matter what kind of light sheet isused, the light sheet either becomes thicker or the illumination lightis less confined as its length increases due to the diffraction oflight. As a result, the size of SPIM FOV always goes opposite to theSPIM axial resolution and optical sectioning capability. Therefore, therelationship between SPIM axial resolution, optical sectioningcapability and FOV represents a key tradeoff in SPIM, and this tradeoffcannot be overcome by regular SPIM due to the diffraction of light.TLS-SPIM provides a solution to this problem.

Real-Time Optimization of the Excitation Light Sheet:

Because of the tradeoff in SPIM, no light sheet is suited for allspecimens. Optimization of the light sheet is required in SPIM, and thebenefit of SPIM is not guaranteed unless an appropriate light sheet isselected based on the specimen to be imaged and the question to beanswered. Different light sheets have been developed for SPIM imaging.Typical light sheets include Gaussian light sheets, Bessel light sheetsand Lattice light sheets, etc [1-7, 15]. For light sheets of comparablesizes, Gaussian light sheets have the most restricted light confinement,but they are usually too thick to obtain submicron axial resolution.Bessel light sheets contain a much thinner central peak that enableshigh axial resolution to be obtained in theory, but the opticalsectioning capability is sacrificed due to the poor illumination lightconfinement. Lattice light sheets reach a better balance between lightsheet thickness and light confinement compared to the previous two byusing a coherent Bessel beam array, but it is more sensitive to opticalaberration and light scattering. [13, 14]

On the other hand, SPIM lowers the photobleaching and photodamagecompared to conventional microscopy techniques. Obviously, light sheetsconfining more excitation light within the detection depth of focusproduce less photobleaching and photodamage. Nevertheless, thephotodamage of SPIM not only depends on the light sheet intensityprofile, but also on how it is generated. In SPIM, the excitation lightsheet can be either a real one, existing simultaneously across theentire illumination field, or a virtual one, created by scanning anexcitation beam or dithering a beam array. As a consequence, for lightsheets of the same intensity profile, a real light sheet has a lowerpeak intensity than a virtual one created by dithering a beam array orscanning a single excitation beam. Although it hasn't been studiedsystematically due to the complexity of a fair comparison and differentnatures of different specimens, lower excitation peak intensity isusually preferred to decrease the photodamage. Using a light sheet withlower peak intensity should always be considered when photodamagebecomes a major problem [6, 7]. A general guidance of how to choose SPIMlight sheet can find in previous publications [13, 14]. For the abovereasons, to adjust the light sheet in real-time based on the immediatefeedback from the imaging result is important to improve the 3D liveimaging ability of SPIM. We conceived our research and constructed ourmicroscope accordingly.

Microscope Configuration

CW lasers with excitation wavelengths of 488 nm and 561 nm are used forlinear excitation (Coherent, Sapphire LP, 200 mW). Laser beams from bothlasers are expanded to a common 1/e² beam diameter of 1.5 mm andcombined into a single co-linear beam using a LaserMux dichroic beamcombiner. The combined laser beam is sent to an acousto-optical tunablefilter (AOTF, AA Opto-Electronic, nAOTFnC-400.650-TN) used to select oneor more wavelengths and modulate the laser beam intensity. The laserbeam is directed to one of the two separated optical paths after theAOTF and a half wave plate HWP1.

The microscope is operated in two modes by sending the laser beam to thetwo separated optical paths. In the first mode, a binary spatial lightmodulator (SLM), SLM2, conjugated to the rear pupil of the excitationobjective is used to generate a single excitation beam or an incoherentbeam array. A virtual excitation light sheet is generated by scanningthe excitation beam or dithering the excitation beam array using amirror galvanometer. SLM2 is also used to tile the excitation lightsheet by changing the excitation beam/beam array position along the beamdirection. In the second mode, Lattice light sheets are used for SPIMimaging. Two binary SLMs, SLM1 and SLM2 are implemented in sequence, inwhich SLM1 is conjugated to the image focal plane of the excitationobjective and SLM2 is still conjugated to the rear pupil of theexcitation objective. A Lattice light sheet is generated by applying thecorresponding phase maps to both SLM1 and SLM2, and the light sheet wastiled by SLM2 in the same way as that in the first mode.

In the second mode, the laser beam is expanded to 15 mm in verticaldirection by a pair of cylindrical lenses (focal length CL1=25 mm,CL2=250 mm) and sent to SLM1. The SLM assembly consisted of a polarizingbeam splitter cube, a half-wave plate and a 1280×1024 pixel binary SLM(Forth Dimension, SXGA-3DM). The SLM1, conjugated to the excitationobjective image focal plane, is imaged to the second identical binarySLM assembly, SLM2, through lens L5=500 mm. After that, the laser beamis sent to the beam scanning assembly consisted of a pair of galvos(Cambridge Technology, 6215HP-1HB) for laser beam and light sheetscanning. Both galvos are conjugated to SLM2, through relay lensesL6=150 mm, L7=75 mm, and conjugated with each other through relay lensesL8=100 mm, L9=100 mm, and finally conjugated to the rear pupil of theexcitation objective (Nikon, CFI Apo 40×W NIR 0.8 NA) through relaylenses L10=75 mm and L11=150 mm. An optical slit was placed at the focalplane of lens L6, to block the undesired diffraction orders generated bySLM2. The detection objective (Nikon, CFI Apo 40×W NIR 0.8NA), mountedon an objective scan piezo (PI, P-724), is placed orthogonal toexcitation objective. The emitted fluorescence is collected through thedetection objective and imaged onto a sCMOS camera (Hamamatsu, OrcaFlash 4.0) by tube lens L12=225 mm. In the first operation mode, thelaser beam is directed to the other optical path by flip mirrors FM1 andFM2. The laser beam is expanded to 6 mm diameter by relay lenses L3=25mm and L4=100 mm and sent to SLM2 after that. The laser beam followedthe same optical path as that in the other mode after SLM2. Manualoperation is required to switch the microscope between the two modes.The flip mirrors FM1 and FM2 can be replaced by galvos to allow fastswitching between the two operation modes. A wildfield optical path usedto find samples before SPIM imaging is not shown in the schematicdiagram. The control system and hardware are the same as what reportedin the previous publications [5, 6, 13]. The sCMOS camera triggers areused to synchronize the operation of the SLMs, galvos, objective piezoand camera itself.

Microscope Working Principle

The microscope was designed to satisfy two requirements. First, theexcitation light sheet can be tiled quickly within the detection imageplane to enlarge the FOV without affecting the axial resolution and theoptical sectioning capability. Next, both the type and the dimension ofthe light sheet can be changed quickly to optimize the SPIM imagingperformance in real-time.

Tiling of the excitation light sheet can be realized by adding avariable spherical phase map to the excitation light wavefront at aplane conjugated to the excitation objective rear pupil. It can beachieved by using either a SLM, a deformable mirror, or a focus variablelens. We use a binary SLM because it is flexible, easy to control andthe light sheet can be positioned fast and accurately by applyingdifferent phase maps. The applied phase map can be refreshed in lessthan a millisecond (˜3.2K Hz rate), which enables both fast tiling andreal-time optimization of the light sheet. The method of tiling thelight sheet with a binary SLM by applying a spherical phase map wasdescribed in details in our previous publication [17].

The excitation light sheet used in SPIM is usually generated by scanninga single excitation beam or dithering a beam array. The intensityprofile of light sheet is adjusted by changing the beam or the beamarray used to create the light sheet. It can be realized by modifyingthe intensity and phase of the excitation laser beam. For example,circular laser beams of different diameters, corresponding to differentexcitation numerical apertures (NA_(exc)) can be used to create Gaussianbeams of different dimensions. Annular laser beams of different innerand outer diameters, corresponding to different inner and outerexcitation NA (NA_(ID), NA_(OD)), can be used to create different Besselbeams. A metal coated quartz mask containing transmissive patterns ofdifferent geometries is usually used to shape the laser beam intensityprofile and create these beams [5-7, 13, 14]. However, the number ofoptions is limited and realignment is required to switch betweendifferent beams in such a way.

In our microscope, SLM2 is also used to modify the intensity and phaseprofile of the excitation laser beam to adjust the light sheet intensityprofile in real-time besides tile the light sheet. The SLM2 is separatedto two areas, retain area and abandon area. A high frequency binaryphase grating is applied to the abandon area, so that the incident lightfalling on the abandon area is diffracted to different directions fromthe light falling on the retain area. The abandoned light is lost orblocked by the optical slit afterwards. The retained light is allowed topropagate along the designed optical path. For example, the annularamplitude mask used to generate a Bessel beam can be replaced byapplying the phase map to SLM2. It is calculated by multiplying areverse binary amplitude mask, and a high frequency binary phase grating(2 pixels per period). In this way, the laser beam intensity profile canbe modified by adjusting the shape and size of the retain area. Furthermodification can be done by controlling the phase within the retain areaof SLM2. Most excitation beams/light sheets can be generated by thismethod, and we developed specific solutions to generate either a singleGaussian/Bessel beam, an incoherent Gaussian/Bessel beam array or acoherent Bessel beam array in our microscope, so that both Gaussianlight sheets, Bessel light sheets and Lattice light sheets can be usedin our microscope. The details are discussed below.

Single Excitation Beam

The microscope is operated in the first mode, and only SLM2 is used. Theexcitation beam and its dimension are controlled by shape and size ofthe retain area on SLM2. The tiling of the light sheet is realized byapplying a binary spherical phase map to the retain area. Extradiffraction orders are blocked by the optical slit. The details togenerate the binary spherical phase map is discussed in our previouspublication [17]. For example, a Bessel beam (NA_(OD)=0.35,NA_(ID)=0.14) was generated by applying the phase map. The same beam wasmoved to the right side of the previous position by applying the phasemap. A thicker and longer Bessel beam (NA_(OD)=0.2, NA_(ID)=0.05) wasgenerated on the left side of the initial beam position by applying thephase map.

Incoherent Beam Array

The microscope also works in the first mode to generate an incoherentexcitation beam array. A light sheet with lower peak intensity can begenerated by dithering the beam array. An incoherent beam array withbeams of roughly equal intensity can be generated by using a Dammanngrating [25, 26], which is a periodic binary phase pattern. In themeantime, the beam array can be tiled by superimposing a continuousspherical phase map to the Dammam grating. A binary phase map togenerate the beam array at a desired position is obtained by resettingthe pixel values of the superimposed phase map to 0 and π. Valuesbetween 0 and π were set to 0, and values between π and 2π were set toπ. The final binary phase map applied to SLM2 is obtained by replacingthe phase map of the abandon area with the high frequency gratingpattern.

However, this method has several limitations due to the limited SLMpixels. To create a beam array of N beams using a Dammann grating,2×int[(N−1)/4]+2 transition points are required per grating period [26],while the minimum distance between any two adjacent transition pointsmust be larger than one pixel to allow the grating pattern to be appliedto the SLM. In consequence, the pixel number per grating period has tobe increased as the number of beams increases, which decreases thecorresponding beam array period at the same time. On the other hand, thebeam array period has to be large enough to make the beam array wideenough to cover the desired FOV and avoid the interference betweendifferent beams, because the Dammann grating doesn't control the phaserelationship between different beams. For above reasons, it is difficultto generate a beam array of more than 21 beams by this method, and theselection of beam array period corresponding to each beam number is alsolimited.

Coherent Bessel Beam Array

TLS-SPIM works in the second mode to use a tiling Lattice light sheet.Both SLM1 and SLM2 are used to generate and tile a coherent Bessel beamarray in this mode. As reported in previous research from Betzig's lab[7], a binary phase map generated from the desired beam array amplitudeand phase profile is applied to a binary SLM that conjugated to theimage focal plane of the excitation objective. The SLM is imaged to atransmissive annular pattern on a photo mask, which is conjugated to theexcitation objective rear pupil to remove the undesired diffractionorders. A similar method is used in our microscope to generate acoherent Bessel beam array except that SLM2 is used to replace the photomask and tile the Lattice light sheet. A binary phase map is applied toSLM1 to generate a coherent excitation beam array. The high frequencygrating pattern is also used to remove the undesired illumination light.Two phase maps applied to SLM2 (NA_(OD)=0.35, NA_(ID)=0.14) to shape theexcitation laser beam and tile the coherent Bessel beam array.

A beam array that is more close to an incoherent beam array can also begenerated in this mode with the same method by increasing the beam arrayperiod. The beam array period and the number of beams can be controlledwith more flexibility in this mode compared to the previous one, despitethe more complicated optical system and more laser power loss due to theuse of two SLMs.

Microscope Alignment and Calibration

The microscope alignment is similar to what reported in our previouspublication [13]. The binary phase maps used to generate differentexcitation beams/beam arrays at different positions in the two workingmodes were calculated in MATLAB in advance and calibrated in fluorescentdye solution. All phase maps were loaded into the corresponding SLMprior to experiments. Selected phase maps are loaded in sequence andsynchronized by the sCMOS camera during 3D imaging. Potentially, phasemaps corresponding to different light sheets can be calculated in-situwith the given parameters of the optical system components after thecalibration.

Image Analysis

3D image stacks collected at different light sheet tiling positions werestitched together according to the length and tiling positions of thelight sheet. The combined image stack was deconvolved using theRichardson-Lucy 3D deconvolution algorithm in MATLAB [13]. The pointspread function (PSF) corresponding to the light sheet was collected byimaging an isolated 100 nm fluorescent particle. The deconvolved resultwas filtered with low pass filters to remove noises beyond themicroscope OTF. Cell segmentation was performed using Matlab and Amira.Analyzed image data were rendered in 3D with Amira for display. Moreaccurate image registration method to combine different image stacks isunder development.

Sample Preparation

C. elegans embryos were dissected from adult worms. Dissected C. elegansembryos were placed directly on top of poly-D-lysine-coated coverslipsfor imaging. The following strains were used: OD95 1tIs37 [pAA64;pie-1::mCherry::HIS-58+unc-119(+)] IV. 1tIs38 [pAA1;pie-1:GFP::PLC∂^(PH)+unc-119(+)], LP162 nmy-2(cp13[nmy-2::gfp+LoxP]) I.RW10029 zuIs178 [his-72(1 kb 5′ UTR)::his-72::SRPVAT::GFP::his-72 (1 KB3′ UTR)+5.7 kb XbaI−HindIII unc-119(+)]. stIs10024[pie-1::H2B::GFP::pie-1 3′ UTR+unc-119(+)].

Nuclei of zebrafish embryos were labeled by ubiquitous expression ofnuclear localized Kikume green/red photoconvertible protein (NLS-KikGR).Membrane labeling was achieved by injecting 25 picograms of a reporterplasmid at the 1-cell stage. The plasmid consisted of a 1 kb fragment ofthe ntla promoter driving expression of membrane localized Cherry, inorder to enrich for expression in mesodermal cells. Injected plasmid DNAis inherited mosaically in zebrafish embryos, causing cells to bescatter-labeled. Embryos were imaged in their chorions at the 6-12somite stages, focusing on the posteriorly localized embryonic structureof the tailbud. To mount the zebrafish embryo, a drop of 1% low meltingtemperature agarose gel was dropped on top of a cleaned coverslip, andthe zebrafish embryo was place on top of the melted Agarose dropafterwards. The embryo is ready for imaging after the agarose gel issolidified. All embryos were imaged in water at 20° C.

The invention is not limited to the disclosed preferred embodiments, andshould be construed to cover all such alternatives, modifications andequivalents as defined in the appended claims.

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What is claimed is:
 1. A method of three-dimensional (3D) multi-cellularspecimen imaging, the method comprising: generating, by a binary spatiallight modulator (SLM), at least one of a single excitation beam and anincoherent beam array conjugated to a rear pupil of an excitationobjective; generating, using a mirror galvanometer, a light sheet by oneof scanning the single excitation beam and dithering the incoherent beamarray; tiling the light sheet for selective plane illumination toenlarge a field of view (FOV) of selective plane illumination microscopy(SPIM); and adjusting, by applying a binary phase map, a dimension andan intensity of the light sheet, wherein both spatial resolution andoptical sectioning capability are maintained within the FOV, and whereinthe FOV is larger than the light sheet.
 2. The method of claim 1,wherein the light sheet is tiled by changing a position of one of thesingle excitation beam and the incoherent beam array.
 3. The method ofclaim 2, further comprising sequentially implementing at least twobinary SLMs, wherein a first SLM of the at least two binary SLMs isconjugated to an image focal plane of an excitation objective, and asecond SLM of the at least two binary SLMs is conjugated to the rearpupil of the excitation objective.
 4. The method of claim 3, furthercomprising generating a lattice light sheet by applying at least onerespective phase map to the first SLM and the second SLM.